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597
Development 126, 597-606 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
DEV8566
A gp330/megalin-related protein is required in the major epidermis of
Caenorhabditis elegans for completion of molting
John Yochem1,2,3,*, Simon Tuck2, Iva Greenwald3,‡ and Min Han1,4
1Department
of Molecular, Cellular and Developmental Biology, 4Howard Hughes Medical Institute, University of Colorado,
Boulder, CO 80309 USA
2Umeå Center for Molecular Pathology, Umeå University, S-901 87 Umeå, Sweden
3Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
‡Present
address: Department of Biochemistry and Molecular Biophysics, Howard Hughes Medical Institute, Columbia University College of Physicians and Surgeons,
New York, NY 10032, USA
*Author for correspondence (e-mail: [email protected])
Accepted 18 November 1998; published on WWW 7 January 1999
SUMMARY
A genetic analysis of a gp330/megalin-related protein,
LRP-1, has been undertaken in Caenorhabditis elegans.
Consistent with megalin’s being essential for development
of mice, likely null mutations reveal that this large member
of the low density lipoprotein receptor family is also
essential for growth and development of this nematode. The
mutations confer a striking defect, an inability to shed and
degrade all of the old cuticle at each of the larval molts. The
mutations also cause an arrest of growth usually at the molt
from the third to the fourth larval stage. Genetic mosaic
analysis suggests that the lrp-1 gene functions in the major
epidermal syncytium hyp7, a polarized epithelium that
secretes cuticle from its apical surface. Staining of whole
mounts with specific monoclonal antibodies reveals that the
protein is expressed on the apical surface of hyp7. Sterol
starvation can phenocopy the lrp-1 mutations, suggesting
that LRP-1 is a receptor for sterols that must be
endocytosed by hyp7. These observations indicate that
LRP-1 is related to megalin not only structurally but also
functionally.
INTRODUCTION
a null mutation of the gene. They die soon after birth with
malformations of the skull (a holoproencephalic phenotype)
that resemble, but are weaker than, those that result when the
biosynthesis of cholesterol is perturbed either by genetic
disease or by the application of certain teratogens (Willnow et
al., 1996). Although these observations suggest a major role
for megalin in the uptake of cholesterol, the genetic analysis
has also suggested a role in regulation of proteases: megalindeficient lungs have malformed alveolar sacs that resemble
those associated with emphysema or with an experimental
perturbation of proteolysis (Willnow et al., 1996). The precise
role, if any, of megalin in the regulation of extracellular
proteases remains unknown, however.
Like mammals, the free-living nematode Caenorhabditis
elegans has at least two genes that can encode > 500 kDa
members of the LDL receptor family. One of these has recently
been revealed by the genome sequencing consortium
(Waterston and Sulston, 1995). Although sharing an overall
structure with megalin and to a slightly greater extent LRP, its
predicted product is unusual (unpublished observations). The
second gene has been called lrp-1 (Yochem and Greenwald,
1993), but its predicted product was later discovered to be more
closely related to rat gp330/megalin than to LRP (Saito et al.,
1994), suggesting that worm LRP-1 and mammalian megalin
In mammals, megalin (also called gp330 or LRP-2, for the
second low density lipoprotein (LDL) receptor-related protein)
is one of two > 500 kDa members of the LDL receptor family
of integral membrane proteins (Saito et al., 1994). Megalin is
expressed on the apical surface of certain epithelia and is
thought to be a clearance receptor that maintains lipid
homeostasis and regulates the activity of extracellular
proteases (for review, see Krieger and Herz, 1994; Strickland
et al., 1995). It can bind, in vitro, a variety of particles and
molecules, including proteases, complexes of proteases and
their inhibitors, and lipoprotein particles. If endocytosed from
extracellular fluids, the ligands are often degraded following
dissociation from the receptor, which is then returned to the
cell surface. Although LRP, the other large member of the LDL
receptor family, is not expressed on the apical surface of
epithelia, it is also an endocytotic receptor and can bind many
of the same ligands as megalin (Krieger and Herz, 1994).
Megalin has been shown to endocytose LDL particles
themselves, suggesting that it may be a high-capacity receptor
that is used by certain tissues that require large amounts of
cholesterol (Stefansson et al., 1995). This proposal is
consistent with the phenotype of mice that are homozygous for
Key words: gp330, Megalin, LRP-2, Sterols, Caenorhabditis
elegans, Molting
598
J. Yochem and others
might be counterparts. The apparently early appearance of
these large proteins in metazoan evolution suggests that they
have a fundamental function that might be revealed by genetic
analysis in a simple animal. In order to investigate this
possibility, likely null mutations have been isolated in the lrp1 gene, revealing that the megalin-related protein is essential
for growth and development of C. elegans. The phenotype of
the mutations is discussed with respect to the pattern of
expression of the gene.
MATERIALS AND METHODS
Genetic markers and media
N2 variety Bristol was the wild-type strain, and the following genetic
deficiencies or recessive mutations were used: Linkage Group (LG)
I: unc-13(e1091) (Waterston and Brenner, 1978), gld-1(q266) (Francis
et al., 1995), ozDf5 (Francis et al., 1995), nDf24 (Ferguson and
Horvitz, 1985); LGIV: him-8(e1489) (Hodgkin et al., 1979).
Chromosomal rearrangements included szT1, a reciprocal
translocation between LGI and LGX (Fodor and Deàk, 1985; Edgley
et al., 1995); and nDp4, a duplication of a large segment of LGI that
is attached to LGV (McKim et al., 1992). nDp4 should contain the
wild-type lrp-1 locus and is largely maintained in the heterozygous
state. If not otherwise indicated, strains were propagated as described
by Brenner (1974) on nematode growth medium (NGM) that
contained a supplement of 5 µg/ml cholesterol, either DifcoBacto or
Sigma type A7002 agar, and lawns of the food source, Escherichia
coli strain OP50. Sterol-deficient medium was similar to NGM but
had the following alterations: bactopeptone was eliminated, agarose
(SeaKem GTG) replaced agar, and cholesterol was not added. Control
medium was identical except for the presence of 50 µg/ml of
cholesterol (Sigma; C-8667; >99% pure) added from a stock solution
(5 mg/ml in 95% ethanol; an equivalent volume of 95% ethanol
lacking cholesterol was added to the sterol-deficient medium). Before
being applied to either medium, overnight cultures of the OP50
bacteria were washed twice with M9 buffer (Brenner, 1974) and then
concentrated 20-fold in M9 buffer.
Isolation of the ku156 and ku157 alleles of lrp-1
BW1707 (provided by J. A. Powell-Coffman and W. Wood,
University of Colorado), which has the genotype unc13(e1091)/nDf24 (I); him-8(e1489) (IV), was the parental strain of the
ku156 and ku157 alleles of lrp-1. Its use was based on the physical
proximity of lrp-1 to unc-13 (A. Coulson, personal communication;
Yochem and Greenwald, 1993) and the assumption that lrp-1 would
be essential for viability or fertility because its product is conserved
with mammalian megalin. Based on previously described methods
(Rosenbluth et al., 1985), BW1707 was treated with gamma rays
(1900 or 3800 rads), and fully coordinated first-generation larvae were
placed one to a plate so that the progeny of each could be examined
for the presence of sterile Unc-13 segregants or for the absence of
Unc-13 segregants, indicating a lethal mutation closely linked to unc13(e1091) (Fig. 1).
Heterozygous males were crossed to BS585 (provided by T. Schedl,
Washington University, St. Louis), a strain having the genotype unc13(e51) ozDf5 I; nDp4(I,V)/+, in order to test complementation
between candidate mutations and ozDf5, a small deficiency that
deletes genes to the right of unc-13 including lrp-1 DNA (Francis et
al., 1995). Mutations that failed to complement and that could
therefore be within the lrp-1 gene were next tested for their ability to
be rescued by an integrated array (kuIs18) that is composed of rol6(su1006) DNA and two overlapping cosmid clones (F29D11 and
C27G12) that contain the lrp-1 gene (A. Coulson, personal
communication).
From a set of 10,000 F1 progeny, six essential mutations were
isolated that could not be complemented by ozDf5. Of these, three
could be complemented by kuIs18. DNA was isolated from balanced
strains containing these three mutations and from the parental strain,
and Southern blots were probed for alterations within lrp-1. The two
mutations in lrp-1 that were isolated have been designated ku156 and
ku157. Each was backcrossed seven times to the N2 strain before
further analyses. The ku156 mutation was separated from the unc-13
mutation by identifying a spontaneous recombinant. The rarity of the
recombination (1/1296) is consistent with the measured linkage of
unc-13 and gld-1, the nearest marker to the right of lrp-1 (Francis et
al., 1995).
Transformation rescue of lrp-1 mutations
Extrachromosomal arrays were formed in vivo following
microinjection of cloned DNA into the syncytial germ line (Mello and
Fire, 1995). The plasmid pRF4 (rol-6(su1006dm)) (Mello and Fire,
1995) was used at a concentration of 100 µg/ml as a dominant marker
for transformation. For construction of MH#jy209b, a plasmid
specific for the 5′ part of the lrp-1 gene, a 16.5 kb SmaI/HpaI DNA
fragment (Fig. 2) was isolated from the cosmid F29D11. For
MH#jy207b, a plasmid specific for the 3′ part, a 14 kb SpeI/SalI DNA
fragment (Fig. 2) was isolated from the same cosmid. The fragments
were inserted into BlueScript vectors, and the resulting plasmids were
each injected at 2.5 µg/ml. Because the plasmids share 2 kb of DNA
from lrp-1, homologous recombination within this region can create
a gene of full length when the plasmids are injected together (Mello
and Fire, 1995). Rescue of ku156 or ku157 was assessed by injecting
MH204 (genotype unc-13(e1091) lrp-1(ku156); nDp4/+) or MH972
(genotype unc-13(e1091) lrp-1(ku157); nDp4/+) with a mixture of the
DNA clones and then determining whether transgenic lines, based on
rolling behavior conferred by pRF4, could produce healthy Unc-13
progeny that could themselves establish stable lines. Rescue of szT1
by lrp-1 DNA was determined by genetic crosses employing
extrachromosomal arrays formed from the injections of MH204 or
MH972. Control injections lacking lrp-1 DNA but containing pRF4
confirmed specificity of the rescue.
Genetic mosaic analysis
Based on the approach of Lackner et al. (1994), spontaneous mosaics
were generated in the progeny of a strain, MH1066, having the
genotype lrp-1(ku156); him-8(e1489); kuEx76[SUR-5GFP(NLS); lrp1(+)]. The extrachromosomal array kuEx76 was formed in vivo
following microinjection (Mello and Fire, 1995) of three DNA clones
into the syncytial germline of a heterozygote balanced for ku156.
F29D11 and C27G12, the two overlapping cosmid clones that contain
genomic DNA from the lrp-1 region, were injected at 2.5 µg/ml each
for rescue of chromosomal copies of lrp-1(ku156). (Subsequent work
demonstrated that the C27G12 clone is superfluous for rescue of lrp1 mutations.) The establishment of trangenic lines and the detection
of mosaicism were based on injecting pTG96 at 100 µg/ml for
expression of SUR-5(GFP)NLS (Yochem et al., 1998), a marker that
expresses a form of the green fluorescent protein (GFP) in many
nuclei. Normal growth plates containing MH1066 were first examined
at magnifications of 40-100× with a dissecting microscope equipped
with a mercury lamp for vigorous L3 and L4 larvae or young adults
that had mosaic patterns of green fluorescence. Using 1000×
magnification, mosaicism was assessed by examining certain nuclei
for the presence or absence of fluorescence as previously described
(Yochem et al., 1998). The mosaics were then examined with
Nomarski optics for the presence or absence of all aspects of the Lrp1 mutant phenotype.
Preparation of monoclonal antibodies against LRP-1
Because the LRP-1/megalin protein is largely composed of many
copies of motifs that are also present in a number of other proteins,
the carboxyl-most region was used for the production of monoclonal
Megalin mutations affect molting
Indirect immunofluorescence
Mixed-stage populations of worms were fixed and made permeable to
antibodies as previously described (Bettinger et al., 1996). A 1:1000
dilution of an ascites preparation of the mouse monoclonal antibody
MH27 was used for detection of adherens junctions between epithelia
(Waterston, 1988; Podbilewicz and White, 1994). Non-ascites
preparations of 1H6 (1:200) and 4H5 (1:150) were used together for
enhancement of the LRP-1 staining. Binding of the antibodies was
detected with a 1:1600 dilution of a Cy3-conjugated antibody against
mouse IgG (Jackson ImmunoResearch). Incubations (16 hours at 4°C)
and washes (20°C over the course of several hours) were as previously
described (Finney and Ruvkun, 1990). Whole mounts were examined
at 1000×, and micrographs were generated with a CCD camera and
Adobe Photoshop.
unc-29
gld-1
lrp-1
goa-1
0.2 map units
unc-13
antibodies in an effort to minimize the chances for cross-reactivity. A
700 bp EcoRI fragment from a cDNA clone encoding the final amino
acids, the stop codon, and 3′ untranslated DNA was cloned into the
vector pGEX-1 (Smith and Johnson, 1988) such that the final 117
amino acids of LRP-1 are fused to the carboxyl terminus of
glutathione S-transferase (GST). The fusion protein was produced in
bacteria and isolated by its binding to glutathione-conjugated beads.
Injections of mice and production of hybridomas were performed by
M. Marlow at the Monoclonal Antibody Facility at Princeton
University. In order to avoid antibodies against GST, supernatants
were assayed by western blotting for reactivity to a fusion protein
containing the same moiety of LRP-1 and β-galactosidase in place of
GST. Two independent monoclonal antibodies, 1H6 and 4H5, were
used for staining whole mounts.
599
nDp4
ozDf5
nDf24
Step 1. Screen for lethal mutations (m) linked to unc-13:
γ-ray
unc-13
+
unc-13
+
+
nDf24
segregates viable
unc-13 homozygotes
m
+
+
nDf24
cannot segregate viable
unc-13 homozygotes
Step 2. Test if m is located within the interval that is deleted in ozDf5:
unc-13 m
+
+
+
么
nDf24
unc-13
unc-13
x
unc-13
unc-13
ozDf5 ;
ozDf5
nDp4
+
么
乆
m
ozDf5
Inviable Unc-13 segregants indicate a potential mutation in the lrp-1 gene
(if viable Unc-13 animals are present, then m is not located with the
ozDf5 interval and therefore cannot be a mutation in lrp-1)
Step 3. Test whether cosmids containing wild-type lrp-1 DNA can rescue the
RESULTS
The lrp-1 gene is essential for growth
Two recessive mutations, ku156 and ku157, have been isolated
in the lrp-1 gene following gamma-irradiation of a strain,
BW1707, that is balanced for mutations linked to unc-13 (Fig.
1). The mutations fail to complement each other and confer the
same phenotype. Homozygotes segregating from heterozygous
mothers hatch and initiate growth and development. Most,
however, lack vigor, and an arrest of growth and development
usually ensues at the molt from the third (L3) to the fourth (L4)
larval stage if not earlier. The homozygotes are moderately
Dumpy (short and fat), have darkened intestines, are small for
their developmental state, and are often encased in old cuticle
or have pieces of old cuticle that remain attached to parts of
the epidermis. Nomarski observations reveal an undefined but
unhealthy appearance, and there is an accumulation of
refractile material in the rectum. Lastly, strains with simple
genotypes such as MH210 (lrp-1(ku156)/gld-1(q266)) fail to
segregate dead embryos, demonstrating that lrp-1
homozygotes are able to complete embryonic development
(and usually early larval development) when they segregate
from heterozygous mothers.
Genetic, molecular, and immunocytochemical data prove
that these are mutations in lrp-1 and suggest that this
description is likely to be the null phenotype. One indication
that these are mutations in lrp-1 is the ability to rescue the
phenotype of either mutation completely following coinjection of two plasmids, MH#jy207b and MH#jy209b, that
contain overlapping subclones of genomic DNA that should
derive only from the lrp-1 locus (Fig. 2). More importantly, the
ku157 mutation is associated with an alteration of the lrp-1
coding region when restricted DNA is analyzed by Southern
inviable mutations that are within the ozDf5 interval
Fig. 1. A screen for lethal mutations in the ozDf5 region of Linkage
Group I. Approximate locations of the genetic deficiencies ozDf5 and
nDf24 are indicated with black bars (Francis et al., 1995). Although
ozDf5 was isolated in cis to unc-13(e51), the speckled region
indicates that the left end of the deletion is not precisely known.
hybridizations: a HindIII fragment migrates more slowly than
the corresponding 5.1 kb fragment of the N2 or BW1707
strains (Fig. 3). Because the flanking HindIII fragments are not
altered, ku157 is an insertion of 0.8 kb of DNA into an internal
region of the lrp-1 gene (Figs 2, 3). Although Southern blot
analysis of EcoRI- or HindIII-digested DNA has failed to
reveal
an
alteration
associated
with
ku156,
immunocytochemical data presented below prove that it is also
a severe mutation in lrp-1. Lastly, genetic tests suggest that
ku156 and ku157 are likely null mutations: animals with the
genotype unc-13(e1091) lrp-1(ku156 or ku157) are similar to
those having the genotype unc-13(e1091) lrp-1(ku156 or
ku157)/unc-13(e51) ozDf5.
A pre-existing mutation in lrp-1 was revealed by serendipity.
In an attempt to balance an unrelated mutation on LGI with
szT1, a reciprocal translocation between LGI and LGX whose
physical breakpoint on LGI maps just to the right of unc-13
(McKim et al., 1988), Dumpy segregants were noticed that had
all of the features of the phenotype described above for ku156
and ku157. Although szT1 homozygotes had been described as
dead embryos (Fodor and Deàk, 1985; Edgley et al., 1995),
similar larvae were seen in three distinct strains containing
szT1, suggesting that these larvae are the segregants that fail to
inherit the untranslocated LGI that normally complements an
essential gene on LGI that is disrupted by the translocation. As
600
J. Yochem and others
expected from the phenotype, genetic tests demonstrate that the
breakpoint is within the lrp-1 gene (unpublished observations).
Briefly, szT1 cannot complement ku156 or ku157, but
extrachromosomal arrays containing the lrp-1-specific
plasmids can rescue the translocated chromosomes. Also,
Southern blot analysis reveals an alteration in a 2.8 kb HindIII
fragment that contains four exons from the lrp-1 gene
(unpublished observations; summarized in Fig. 2). Thus, three
mutations, each likely to be severe, have been isolated in the
lrp-1 gene, revealing that it is essential for growth and
development.
lrp-1 is required for completion of molting
A striking effect seen with ku156, ku157, or szT1 homozygotes
is a failure of many to shed all of the old cuticle when molting
from one larval stage to the next. This failure can result in
worms being completely encased in old cuticle that has become
displaced from the anterior end (and sometimes from the
extreme posterior end) but not from the rest of the body (Fig.
4A). These worms can have one of two fates. Many of them
remain stuck in this cuticle but continue to live for several days.
Others are able to break through the old cuticle, but it or parts
of it remains attached to the body; these worms therefore have
rings of old cuticle that encircle the body or have a trail of old
cuticle that remains attached to the posterior part of the body
just anterior to the anus (Fig. 4B,C). In contrast to the shed
cuticle of wild-type C. elegans, the displaced cuticle of an lrp1 homozygote does not degrade within the lifetime of the worm.
Observations with a dissecting microscope sometimes reveal
Dumpy, nonvigorous segregants that appear not to have old
cuticle associated with them. When examined at high
magnification with Nomarski optics, many of these can be seen
to have small pieces of old cuticle that remained attached to
their bodies. Others, however, do not have old cuticle
associated with them. If allowed to resume growth, they arrest
by the time of the L3 to L4 molt and have become encased in
old cuticle or have trails of old cuticle.
Sal I
Hpa I
Hpa I
Hpa I
Hpa I
Hpa I
Spe I
Spe I
Sal I
Spe I
Sal I
Sma I
Fig. 2. A schematic
rat
representation of the lrp-1 gene
C. elegans
and its predicted product. The
coding region (Yochem and
Greenwald, 1993) is indicated in
AATAAA
23
25
relation to 30 kb of DNA from
the cosmid clone F29D11, the
complete sequence of which has
3'-end of
3'-end of
szT1
neighboring gene
been detemined by the C. elegans
neighboring gene
ku157
sequencing consortium and is
probe
available from GenBank or
ACeDB (A C. elegans Data
Base) (Waterston and Sulston,
1995). The true 5′-end of the
F29D11
genomic DNA in the cosmid
16.5 kb Sma I/Hpa I
clone corresponds to the zero
14 kb Spe I/Sal I
position. The 3′-ends of the two
genes predicted to flank lrp-1 are 0
5
10
15
20
25
30 kb
indicated with arrows. The 16.5
kb SmaI/HpaI fragment and the 14 kb SpeI/SalI fragment used for constructing the plasmids MH#jy207b and MH#jy209b are indicated with
brackets below the restriction-enzyme map. The region used as a hybridization probe for the Southern blot shown in Fig. 3B is indicated with a
line, and the HindIII restriction fragments affected by the ku157 or szT1 mutations are represented as bars. The parts of the predicted protein
encoded by exons 23 to 27 are shown because of their relevance to the mutations. The motifs present in LRP-1 and rat gp330/megalin are
indicated as follows: boxes, Class A (ligand binding) repeats; white ovals, class B.2 epidermal growth factor (EGF) repeats, black ovals, class
B.1 EGF repeats; cross-hatched lines, YWTD spacers (Saito et al., 1994).
A comparsion with wild-type molting indicates that the
displaced cuticle of the lrp-1 homozygotes represents old
cuticle that should have been shed and degraded during molts
Fig. 3. Southern blot analysis of ku157 DNA. (A) Hybridization of
radiolabeled F29D11 to HindIII-digested DNA. Lane 1, the wildtype pattern present in DNA from N2 or the parental strain BW1707.
Lane 2, DNA from a strain with the genotype unc-13(e1091); lrp1(ku156); nDp4/+. Lane 3, DNA from a strain with the genotype
unc-13(e1091) lrp-1(ku157); nDp4/+. The wild-type 5.1 kb fragment
and the 5.9 kb fragment associated with ku157 are indicated with
arrows, and the 3.2 and 2.8 kb fragments that flank the 5.1 kb
fragment are indicated with arrowheads. (B) Proof that the altered
5.9 kb band is specific for the lrp-1 coding region was obtained by
removing most of the F29D11 signal and then re-hybridizing the
filter with a probe covering the region of lrp-1 indicated in Fig. 2.
Note that the 5.1 kb fragment hybridizes with half the intensity of the
5.9 kb fragment; most of the progeny should have inherited two
copies of the ku157 allele but only one copy of the wild-type lrp-1
gene because they are heterozygous for the balancer nDp4 (McKim
et al., 1992).
Megalin mutations affect molting
rather than an aberrant blistering of the cuticle of animals not
undergoing molts as is seen with mutations in the six bli genes
(Kramer, 1997). During normal molts (Singh and Sulston,
1978), new cuticle is first synthesized under the old cuticle. Old
cuticle from the anterior half of the pharynx is then expelled,
and old body cuticle with the pharyngeal cuticle attached
separates from the anterior end of the body. That the lrp-1
homozygotes have displaced these cuticles is very evident (Fig.
4A). Moreover, the cuticle of the rectum, the excretory duct,
and regions of the body from which cuticle has not detached
are clearly thicker than normal, suggesting that new cuticle has
been synthesized as for a normal molt.
During normal molts (Singh and Sulston, 1978), the entire
animal rotates within the old cuticle, presumably to loosen it
from the midbody. This is following by a rupturing of the
anterior cuticle, which is associated with contractions of the
head, and emergence of the animal from the old cuticle which
disappears. Like the wild-type worms, lrp-1 homozygotes can
undergo contractions of the head after displacement of the
phayngeal and anterior cuticles. These contractions, however,
appear weak relative to those of wild-type worms, and the
rotation of the entire body is not seen. Taken together, these
observations indicate that the homozygotes initiate the process
of molting but are unable to complete it. In particular, they are
unable to degrade the old cuticle.
Other aspects of the
mutant phenotype
Lrp-1 segregants from szT1
strains or from strains
containing ku156 no longer
in cis to unc-13(e1091) are
usually stationary although
some are capable of moving
a few worm lengths and can
respond appropriately to
harsh touch with a metal
wire or to gentle touch with
a cilium. Occasional worms,
including some that are
completely incased in old
cuticle, can locomote for
several millimeters, but they
eventually become stationary
and usually arrest growth at
the L3 to L4 molt.
Examination
with
a
dissecting
microscope
reveals that the lrp-1
mutations
confer
a
darkening of the intestine.
Because specialized L3
larvae termed dauers also
have darkened intestines
(Riddle and Albert, 1997),
the possibility exists that
the mutant homozygotes
have entered this state
of diapause which is
induced by starvation and
high population densities.
601
Examination with Nomarski optics indicates that this is not
the case. Some of the vesicles in the mutant intestine are
highly refractile and have a necrotic appearance; these vesicles
exactly correspond to dark particles seen with bright-field
illumination. In contrast, the intestines of dauers were seen to
be packed with dense vesicles that lack high refractility.
Furthermore, alae and a constricted pharynx, both
morphological features of dauers (Riddle and Albert, 1997),
are absent in the lrp-1 homozygotes. Although not resembling
the intestines of dauers, the mutant intestines do resemble
those of wild-type worms that have been starved for bacteria
but have not entered the dauer state. Thus, the darkened
intestine may be a consequence of not feeding well, especially
for homozygotes whose mouths are obstructed by undegraded
cuticle.
Nomarski observations of szT1, ku156, or ku157
homozygotes reveal an accumulation of refractile material in
the rectum in those animals that have not shed the old cuticle
from this region (Figs 4, 5). The rectal hypodermal cells K.a
and K′, which produce some of the rectal cuticle (Sulston et
al., 1983), are enlarged and have a refractile sphere – perhaps
a Golgi apparatus – near their nuclei (Fig. 5). There is often a
posterior displacement of the nuclei of the rectal cells B and F
and of other cells in the tail that perhaps results from the
enlargement of K.a and K′.
Fig. 4. Undegraded cuticle is associated with mutations in lrp-1. Longitudinal views with Nomarski optics,
dorsal side up and anterior usually to the left (as is also the case for all subsequent micrographs). (A) An
szT1 homozygote at the L2 stage that had been stuck for several hours in old cuticle. Expulsion of the
pharyngeal cuticle (arrow) is evident. (B) Old cuticle (arrow) that remained attached to the posterior of an
Lrp-1 segregant, from the strain MH210, that was arrested at the molt to the L4 stage. Refractile material in
the rectum can also be seen (arrowhead). (C) Cuticle that remained attached to the tail of an L3 larva from
MH210 had become filled with excrement, emphasizing the enduring nature of the old cuticle. (D) Growth
of the wild-type N2 strain on medium deficient in sterols resulted in a young L3 larva having refractility of
the rectum (arrowhead) and a displaced anterior and pharyngeal cuticles (arrow). Scale bars (A,B,D)
20 µm; (C) 100 µm.
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J. Yochem and others
zygote
AB
P1
ABp
ABa
ABal
EMS
ABar
ABpl
ABpr
ABara ABarp
ABpla ABplp
hyp7
hyp7
hyp7
MS
P2
E
C
Ca
P
3
Cp
D
P4
hyp7 hyp7
Fig. 6. Parts of the early cell lineage in which inheritance of wildtype lrp-1 DNA can correct the mutant phenotype. Data for 15
mosaics that lacked features of the Lrp-1 mutant phenotype are
summarized schematically with respect to the early cell lineage and
the early progenitors of the hyp7 syncytium (Sulston et al., 1983).
Each of the 14 squares represents one animal and depicts the only
cell that established a genotypically wild-type clone of cells in that
animal. An additional animal had two wild-type clones (circles), one
of which (ABal) should not have contributed to the hyp7 syncytium.
Fig. 5. lrp-1 affects the rectum. (A) Nomarski view of an enlarged K′
cell (arrow) and of refractile material in the rectum (arrowhead) of a
mid-L3 larva from MH210. (B) A wild-type rectum is present in a
non-mutant L3 larva from MH210. Scale bars, 5 µm.
Mosaic analysis suggests that the epidermal
syncytium hyp7 is the focus of lrp-1 activity
An analysis of mosaics, individuals having both genotypically
wild-type and genotypically mutant cells (Herman, 1995),
permitted a genetic assessment of the cells that require the
function of lrp-1. Spontaneous mosaics in which an
extrachromosomal array containing wild-type lrp-1 DNA
failed to disjoin during embryogenesis, were identified among
the progeny of a strain, MH1066, having the genotype lrp1(ku156); him-8(e1489); kuEx76[SUR-5GFP(NLS); lrp-1(+)].
Inheritance of the array by AB, P1, ABarp, ABpla, P2, or Ca
is sufficient for rescue of all aspects of the mutant phenotype
(Fig. 6). In particular, the rectal defects are corrected even if
the rectal cells themselves fail to inherit the array. Although
other arguments can be put forth (for instance, inheritance of
lrp-1 activity by any cell might be sufficient), inheritance by
hyp7 is the only lineal commonality of the mosaics, suggesting
that this syncytium, which is formed by the fusion of cells that
descend from ABa, ABp, and C (a granddaughter of P1)
(Sulston et al., 1983), is the focus of the gene’s activity (see
also Herman and Hedgecock, 1990).
A maternal contribution of wild-type lrp-1 activity could
explain the ability of most lrp-1 homozygotes that segregate
from heterozygous mothers to develop to the L3 to L4 molt.
Furthermore, a member of the LDL receptor family might be
expected to function in the endocytosis of yolk proteins by
oocytes. In order to test for a maternal contribution, mosaics
in which the germ line did not inherit the kuEx76 array were
examined for the production of progeny. In all four cases,
progeny were produced, and they exhibited a phenotype
indistinguishable from that of the homozygotes segregating
from heterozygotes. Thus, activity of lrp-1 is not absolutely
required for endocytosis of yolk proteins, nor is it absolutely
required for embryonic development. An incompletely
expressed effect on embryogenesis cannot be excluded,
however, because MH1066 contains a him-8 mutation that
results in death of embryos (Hodgkin et al., 1979), and these
could have masked embryonic lethality caused by the germline mosaicism.
LRP-1 is present on the apical surface of hyp7
By means of monoclonal antibodies that had been produced
against the carboxyl terminus, expression of the lrp-1 gene
product was investigated in mixed-stage populations of N2
worms using conditions (Bettinger et al., 1996) in which 99%
of whole mounts stained with both anti-LRP-1 antibodies and
MH27. Most of the staining is punctate, has an appearance
consistent with circular or spheroidal aggregates of the
protein, and is evident only in hyp6 and hyp7 (Fig. 7), two
syncytia that compose most of the epidermis (White, 1988).
Co-staining with MH27, an antibody that recognizes adherens
junctions (Waterston, 1988; Podbilewicz and White, 1994),
demonstrates that the staining is confined to the dorsal and
ventral ridges and to wider sections that are adjacent to the
lateral seam cells, which themselves fail to stain. Therefore,
the regions of staining correspond to the thick regions of this
epidermis (with respect to a cross-sectional view of worms);
staining is absent (Fig. 7) from longitudinal bands that
correspond to the thin regions of hyp7 to which body muscles
attach (White, 1988). The staining is evident in both sexes at
hatching, in each of the four larval stages, and in adults. The
staining is often brighter in hyp6 in L1 larvae and in the
posterior part of hyp7 in adult males. Wild-type worms that
Megalin mutations affect molting
603
have been starved of bacteria for 4 days also stain, albeit less
failure of lrp-1 mutants to endocytose sterols could explain the
intensely than unstarved worms.
critical nature of the mutant phenotype. Wild-type N2 worms
Staining of segregants from a strain, MH210 (lrpgrowing on medium containing a suboptimal level of sterols
1(ku156)/gld-1(q266)), demonstrated that the epitope(s) of
were therefore examined for any resemblance to the mutant
1H6 and of 4H5 are not present in fixed ku156 homozygotes.
phenotype. After transfer to deficient medium, about 5% of
In contrast to the N2 strain, 26%
of 137 segregants completely
lacked the LRP-1 pattern but did
stain with the MH27 antibody,
demonstrating that they were
fixed
and
permeable
to
antibodies. Thus, the proportion
not staining agrees with the
predictions
of
Mendelian
genetics.
Moreover,
14
segregants
could
be
unambigously identified as lrp-1
homozygotes because they had
refractile material in their recta.
Each of these completely failed
to stain with the anti-LRP-1
antibodies but did stain with
MH27 (Fig. 7). The 1H6 and
4H5 monoclonal antibodies are
therefore specific for the LRP-1
protein, and the absence of
staining of the homozygotes
suggests that ku156 is a likely
null mutation, in agreement with
the genetic tests.
Because MH27 recognizes an
epitope associated with zonulae
adherens that form at the apical
junctions of polarized epithelia
such as hyp6, hyp7, and the
lateral seam cells (Waterston,
1988; Podbilewicz and White,
1994), a final conclusion is
possible: location of >90% of the
LRP-1 staining to the same focal
Fig. 7. Expression of the lrp-1 gene product in apical regions of hyp7. Each specimen has been
plane as the MH27 staining
stained for LRP-1 with the 1H6 and 4H5 monoclonal antibodies. Except for B, each has also been
demonstrates that LRP-1 is
stained for adherens junctions with MH27. All fluorescence micrographs represent the extreme left
located in the apical region of
focal plane. (A) Bands of apical, punctate staining of LRP-1 are present along the entire length of
hyp6 and hyp7 (Fig. 7). In
hyp7 (only an anterior section is shown) in an adult N2 hemaphrodite. Two wide bands of the LRP-1
particular,
the
basolateral
staining (large bracket) flank MH27 staining of the boundary between hyp7 and the lateral seam
surfaces are never seen to stain.
syncytium (arrows) (compare with E for an MH27 pattern in the absence of the LRP-1 pattern). A
Thus, the resemblance between
small bracket depicts the band of LRP-1 staining in the ventral ridge of hyp7; also shown is MH27
LRP-1 and mammalian megalin
staining of the junction between the excretory pore and hyp7 (arrowhead). Note the absence of
extends to their localization at
staining in the zone to which body muscles attach (between the brackets). (B) The LRP-1 pattern in
the apical surface of polarized
the midbody of an N2 hermaphrodite in the absence of staining with MH27. The circular nature of the
punctate staining is particularly evident (thin lines). Based on staining of the dorsal and ventral ridges,
epithelia.
Sterol starvation can
phenocopy lrp-1 mutations
The resemblance of LRP-1 to
mammalian megalin suggests
that
the
worm
protein
endocytoses
sterols
like
cholesterol. Because sterols are
an essential nutrient for C.
elegans (Chitwood, 1992), a
in focal planes not shown, the central zone (between arrowheads) that lacks staining corresponds to
lateral seam cells. (C) LRP-1 staining in hyp7 on either side of the lateral seam in the midbody of a
non-mutant L2 segregant from MH210. Co-staining with MH27 reveals the apical junctions of the
lateral seam, two cells of which are indicated (arrows). LRP-1 staining in the ventral ridge of hyp7 is
also evident (arrowhead). (D) An interior view with Nomarski optics of a more posterior region of the
same animal as in C, demonstrating a lack of refractile material in the rectum. (E) A mutant L2
segregant of MH210 lacks LRP-1 staining. MH27 staining of the apical junctions between hyp7 and
the lateral seam cells, one of which is indicated with an arrow, demonstrates that the specimen was
permeable to antibodies. (F) An interior view with Nomarski optics of a more posterior region of the
same animal as in E, revealing refractile material in the rectum, indicating homozygosity of the lrp-1
mutation. Scale bars (A,B) 20 µm; (C-F) same scale as A.
604
J. Yochem and others
larvae of the second generation but none of the first generation
were Dumpy, had problems shedding old cuticle during molts,
and sometimes had refractile material in their recta (Fig. 4D).
In addition, some adults of the second generation exhibited an
abnormal loopy movement of their bodies and were unable to
move effectively. (Similar behavior is occasionally displayed
by adult segregants of transgenic lines in which the szT1 or
ku156 mutations are rescued with extrachromosomal arrays.
This congruence may indicate that the mutations were
incompletely rescued by the array in these animals.) In
contrast, N2 worms that had been grown on otherwise identical
medium that had been supplemented with 50 µg/ml cholesterol
lacked all of these defects. Although the starvation was not
absolute (worms continued to propagate at half the rate of
control populations on cholesterol-supplemented medium) and
two generations were required before effects were evident, the
ability of the starvation to cause the same defects as the Lrp-1
mutant phenotype leaves open the possibility that LRP-1
endocytoses sterols.
DISCUSSION
Genetic, molecular, and immunocytochemical data indicate
that null mutations have been isolated in the lrp-1 gene. A
striking effect of these mutations is an inability to shed and
degrade all of the old cuticle during larval molts. The cuticle
is a collagen-rich exoskeleton that functions as a barrier to the
environment (Kramer, 1997), and an inability to degrade it is
consistent with a perturbation of extracellular proteolysis.
Perhaps the extracellular part of LRP-1 is required for
activiation of collagenase or other proteases that might be
secreted during molts. Alternatively, LRP-1 may be needed for
proteolytic processing of procollagens or other components of
the cuticle. Insufficient maturation of the precursors might
produce a cuticle that is resistent to degradation and lacks
pliability, rendering worms unable to move well. Aberrant
synthesis of the cuticle could also account for the Dumpy
phenotype (Kramer, 1997). Because homozygotes that have
managed to shed most of the old cuticle are also Dumpy, it
might be an intrinsic property of the newly synthesized cuticle.
The presence of LRP-1 on the apical surface of hyp6 and
hyp7 is completely consistent with the failure of the mutants
to shed and degrade old cuticle. The hyp7 syncytium composes
most of the epidermis (also called the hypodermis) of C.
elegans (White, 1988). (Because the much smaller hyp6
syncytium fuses with hyp7 during the L2 to L3 molt (Yochem
et al., 1998), for simplicity only hyp7 will be emphasized.) The
apical plasma membrane of hyp7 is the outermost plasma
membrane for most of the length of the body, and from this
apical membrane is secreted the components for most of the
body cuticle (Kramer, 1997). Before a cycle of endocytosis is
initiated, the large extracellular part of LRP-1 must therefore
be located just beneath the overlying cuticle or in contact with
it. A cycle of endocytosis from the apical surface of hyp7
should also be associated with coated pits and endocytotic
vesicles; in the case of mammalian megalin, the latter are
particularly large multivesicular bodies that have been termed
apical vesicles (Chatelet et al., 1986; Willnow et al., 1996).
Thin-section electron microscopy of C. elegans has revealed
large multivesicular bodies in the syncytial epidermis that are
similar in appearance to apical vesicles (White, 1988);
detection of these vesicles by the anti-LRP-1 antibodies would
be a straight-forward interpretation of the punctate, circular
pattern of staining. Thus, although LRP-1 has not been proved
to be a receptor, the available data suggest that it functions as
such in a location that is consistent with the mutant phenotype
and with the proposal that one function of this class of receptor
is for the regulation of extracellular proteolysis (Krieger and
Herz, 1994; Strickland et al., 1995; Willnow et al., 1996).
Perhaps a more likely explanation for the mutant phenotype,
however, is indicated by the effect of sterol starvation: the fact
that starvation phenocopies the mutations suggests that LRP-1
endocytoses dietary sterols like cholesterol. The presence of
LRP-1 in the apical region of hyp7 may appear at first glance
to be inconsistent with such a function, as the sterols would
have to pass through the cuticle before encountering the
receptor. Experiments with the large parasite Ascaris suum
have, however, indicated that nematodes primarily absorb
sterols through the epidermis rather than through the intestine
(Fleming and Fetterer, 1984). The presence of LRP-1 in the
apical region of a polarized epithelium may therefore serve the
same function as the major one deduced for mammalian
megalin: the endocytosis of cholesterol from extensive fluids
that are external to these epithelia (Willnow et al., 1996).
Although propagation of C. elegans is known to require
dietary sterols (Chitwood, 1992), the morphological effects of
sterol deprivation have not been previously reported. Sterol
starvation of other nematodes has been reported to cause
incomplete molts (Bottjer et al., 1984; Coggins et al., 1985),
but a comparison with the Lrp-1 mutant phenotype is difficult
because the effects on molting were not described in detail. As
was the case described here for C. elegans, sterol starvation of
the free-living nematodes Panagrellus redivivus and C.
briggsae, a close relative of C. elegans, required two
generations before arrest of growth (Hieb and Rothstein, 1968;
Bottjer et al., 1985). This lag is at variance with the
manifestation of the Lrp-1 phenotype in homozygotes as they
segregate from heterozygous mothers. One explanation is that
sterols had not been sufficiently eliminated from the media; it
is difficult to starve nematodes of sterols because
microbiological media are often contaminated with them, and
nematodes can convert plant or yeast sterols to useful forms,
including cholesterol (Chitwood, 1992). It is possible that the
first generation were unaffected because of a combination of a
maternal supply in the eggs from which they arose and an
ability to scavenge contaminating sterols from the medium.
The level of contaminating sterols may have been too low,
however, for an adequate maternal contribution to the next
generation.
Because it is not known why sterols are essential for
nematodes (Chitwood, 1992), interpretation of the effect of the
deprivation on molting remains speculative. Perhaps a lack of
cholesterol in cell membranes renders worms too weak to
complete the process. Also, degradation of old cuticle may be
activated by a developmental signal that requires a cholesterol
modification such as occurs for the Drosophila protein
hedgehog (Porter et al., 1996). Another possiblity is that a
steroid hormone such as 20-hydroxyecdysone regulates
molting in C. elegans. It is interesting to note in this regard that
‘RNA interference’ (Fire et al., 1998) of CHR3, an orphan
nuclear hormone receptor, results in a failure to shed and
Megalin mutations affect molting
degrade all of the old cuticle during molts (Kostrouchova et al.,
1998). CHR3 closely resembles DHR3, a Drosophila protein
that affects metamorphosis and is induced upon treatment with
20-hydroxyecdysone (Lam et al., 1997; White et al., 1997). If
expression of CHR3 also requires ecdysone, a failure of lrp-1
mutants to endocytose sterols could produce the same
phenotype. It is controversial, however, whether normal
molting of nematodes is regulated by ecdysone, and production
of ecdysone from cholesterol has yet to be demonstrated for
any nematode (Chitwood, 1992). By what ever means, the
genetic mosaic analysis of lrp-1 is consistent with signaling
between cells, because the rectal defects were corrected in
mocaics even when the rectal cells themselves failed to inherit
the wild-type gene.
The effects of sterol starvation on nematodes suggests that
LRP-1 endocytoses dietary sterols that are needed for an
unknown function during molting. Nevertheless, the alternative
must be considered that the effects of the starvation arise from
perturbation of a component that acts in parallel to or in
conjuction with LRP-1 during molts. Also, it is not clear why
the structures of LRP-1 and mammalian megalin are so closely
conserved if they are only required for the uptake of sterols.
We have begun a genetic approach to the question of function
by isolating mutations in other genes that confer the same
phenotype. Also, mutations in the genes let-454, let-462, and
let-473 have been mentioned as affecting molting (Johnsen and
Baillie, 1991). The analysis and cloning of these and further
analysis of CHR3 (Kostrouchova et al., 1998) may also help
reveal the process in which LRP-1 functions and why this
process is essential for growth and completion of molting.
We are grateful to Alan Couslon for providing cosmid clones, to
Tim Schedl, Jo Anne Powell-Coffman, and William Wood for
providing strains, to Martha Marlow for her murine and hybridoma
expertise, and to Ross Francis and Robert Waterston for providing the
MH27 antibody. Support was from an NIH grant (GM47869) awarded
to M. H. and an award to S. T. from the Cancerfonden. M. H. and I.
G. are investigators of the Howard Hughes Medical Institute.
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